Class IIA Histone Deacetylase (HDAC) Substrate

ABSTRACT

The present invention relates to the use of trifluoroacetylated lysine (Boc-L-Lys(ε- trifluoroacetyl)-MCA) as a substrate for histone deacetylases, specific for the class Ha subtype.

The present invention relates to a substrate for histone deacetylases, specific for the class IIa subtype that is suitable for use in an assay for screening for HDAC subtype-selective inhibitors.

In eukaryotic cells the orderly packaging of DNA in the nucleus plays an important role in the regulation of gene transcription. Nuclear DNA is ordered in a compact complex called chromatin. The core of the complex is an octamer of highly conserved basic proteins called histones. Two each of histones H2A, H2B, H3 and H4 associate and DNA winds around the basic amino acids of the histones interacting with the negatively charged phosphate groups of the DNA. One molecule of histone H1 is associated with each wound core which accommodates approximately 146 bp of DNA. The cores are, in turn, packaged into a compact regular structure with about 200 bp of DNA between each core.

The amino-terminal tails of the histones are subject to post-translational modification, in particular by acetylation of lysine. Histone deacetylases (HDACs) and histone acetyl transferases (HATs) determine the pattern of histone acetylation, which together with other dynamic sequential post-translational modifications might represent a ‘code’ that can be recognised by non-histone proteins forming complexes involved in the regulation of gene expression. This and the ability of histone deacetylases (HDACs) to also modify non-histonic substrates and participate in multi-protein complexes contributes to the regulation of gene transcription, cell cycle progression and differentiation, genome stability and stress responses.

Eleven members of the Zn-dependent HDAC family have been identified in humans, which share a conserved catalytic domain and are grouped into two classes: class I (1,2,3,8), homologous to yeast Rpd3; class IIa (4, 5, 7, 9) and IIb (6, 10), homologous to yeast Hda1. HDAC11 shares homologies with both classes, but is at the same time distinct from all the other ten subtypes. Interest in these enzymes is growing because HDAC inhibitors (HDACi) are promising therapeutic agents against cancer and other diseases. The first generation of HDACi were discovered from cell-based functional assays and only later identified as HDAC class I/II inhibitors. Present HDAC inhibitors are pan-specific or poorly selective. Those that entered clinical trials all show similar adverse effects, mainly fatigue, anorexia, hematologic and GI-toxicity, that becomes dose-limiting in clinical trials. It is not at all clear whether the antitumor properties of HDAC inhibitors are due to their lack of specificity or are the consequence of hitting one or few “crucial” subtypes. This question is of considerable interest because it may open the way for the development of novel, more sensitive compounds with possibly enhanced efficacy and/or tolerability. More recent studies were therefore directed to better define the biological function of different class members and to devise subtype-selective enzymatic assays to assist in the development of improved cancer chemotherapies.

The class IIa HDACs contain a highly conserved C-terminal catalytic domain (˜420 amino acids) homologous to yHDA1 and an extended N-terminal domain that is able to interact with several, different proteins, including transcription factors, transcriptional co-repressors and 14-3-3 proteins. The activity of the class IIa HDACs is regulated at several levels, including tissue-specific gene expression, recruitment of distinct cofactors and nucleocytoplasmic shuttling. Whereas most class I HDACs are ubiquitously expressed, class IIa HDACs are expressed in a restricted number of cell types.

Riester et al., Biochem. Biophys. Res. Comm., (2004) 324:1116-1123 and Wegener et al, Chem. Biol. (2003) 10:61-68, describe the substrate specificities of different HDACs (rat liver HDAC, HDAC8) towards using short fluorogenic substrates. HDAC from rat liver (which according to immunochemical analysis is a mixture of HDAC1, 2 and 3) was shown to have a very low sequence specificity and was able to work also on a substrate composed only of an acetylated lysine (Boc-L-Lys(ε-acetyl)-MCA; K_(M)=3.7 μM). For (recombinant) human HDAC8, no good substrates were reported at the time of the paper. This was true for an H4-derived peptide, as well as for other synthetic substrates. With Boc-L-Lys(ε-acetyl)-MCA or Ac-Arg-Gly-L-Lys(ε-acetyl)-MCA, for example, a K_(m)≧500 μM was published.

In Riester et al. various modifications of ε-acetyl moieties of L-Lysine were also studied. In particular, Boc-L-Lys(ε-trifluoroacetyl)-MCA proved to be a superior substrate for HDAC8 with K_(M) value (K_(M)=247 μM) significantly improved as compared to Boc-L-Lys(ε-acetyl)-MCA. Rat liver HDAC did not convert Boc-L-Lys(ε-trifluoroacetyl)-MCA to a significant degree.

It has now surprisingly been found that trifluoroacetylated lysine (Boc-L-Lys(ε-trifluoroacetyl)-MCA) is a superior substrate for ClassIIa HDACs.

Thus, in a first aspect of the invention there is provided the use of trifluoroacetylated lysine (Boc-L-Lys(ε-trifluoroacetyl)-MCA) as a substrate for class IIa histone deacetylases.

In another aspect there is provided a complex of trifluoroacetylated lysine (Boc-L-Lys(ε-trifluoroacetyl)-MCA) bound to a Class IIa HDAC.

In another aspect there is provided an assay for determining the concentration of a Class IIa HDAC in a sample which comprises:

-   -   (i) incubating a sample containing a Class IIa HDAC with the         substrate trifluoroacetylated lysine         (Boc-L-Lys(ε-trifluoroacetyl)-MCA);     -   (ii) adding a developer and measuring the concentration by the         fluorescence (ex 360 nM, em 460 nM).

Preferably, the developer is a commercially available kit component (BIOMOL Fluor De Lys) or a proteolytic enzyme such as Trypsin or Lys-C that will cleave the bond between the deacetylated lysine and the MCA fluorophore, thereby liberating a fluorescent compound. This assay is capable of determining the sub- nanomolar concentration of the enzyme.

In another aspect is provided an assay for detecting Class IIa HDAC which comprises:

-   -   (i) incubating a sample potentially containing Class IIa HDAC         with the substrate trifluoroacetylated lysine         (Boc-L-Lys(ε-trifluoroacetyl)-MCA); and     -   (ii) detecting the presence of MCA by its fluorescence.

In an embodiment this assay can be used to specifically detect HDAC 4.

In another aspect there is provided an assay specific for Class IIa HDAC inhibitors, which comprises:

-   -   (i) incubating a potential Class IIa HDAC inhibitor with a         protein which expresses the catalytic domain of Class IIa HDAC,         in a suitable assay buffer;     -   (ii) adding the substrate trifluoroacetylated lysine         (Boc-L-Lys(ε-trifluoroacetyl)-MCA) and a developer and         incubating; and     -   (iii) stopping the incubation and determining the effect the         putative Class IIa HDAC inhibitor has on enzyme activity by         comparison with a standard.

Preferred Class IIa HDACs are HDAC4, HDAC5 and HDAC7, particularly HDAC4.

Preferably, the protein which expresses the catalytic domain of Class IIa HDAC is E coli, particularly strain BL21.

Preferable, the concentration of the Class IIa HDAC enzyme in the assay for screening for inhibitors is 0.05-50 nM preferably 0.5 nM.

Preferably, the assays of the present invention can be performed at a pH value between 6.0 and 8.5, most preferably at pH 7.5. A preferred buffer used for assuring pH stability during the assay is Hepes at a concentration of 20 mM.

In an embodiment, monovalent and/or divalent cations can be added to the assays of the present invention, preferable at millimolar concentrations. Preferred cations are Na, K and Mg.

Preferably, the cations are present in a concentration of 0.5 mM to 200 mM.

Preferred salts which provide the cations are NaCl, KCl and MgCl₂.

In an embodiment NaCl is present at a concentration of 100-150, particularly 137 mM.

In another embodiment KCl is present at a concentration of 0.5-10, particularly 2.7 mM.

In another embodiment MgCl₂ is present in a concentration of 0.5-5, particularly 1 mM.

In an embodiment, a protein such as bovine serum albumin is added to the assay buffer. Preferable, the bovine serum albumin is present at a concentration of 0.01 and 10 mg/ml, particularly at 0.1 mg/ml.

The substrate trifluoroacetylated lysine (Boc-L-Lys(ε-trifluoroacetyl)-MCA) can be added in a concentration range between 1 μM and 100 μM, preferably at 20 or 40 μM

The assays of the present invention can be carried out at a temperature of about 20 to 37° C., preferably about 37° C.

In a preferred embodiment is an assay specific for Class IIa HDAC inhibitors, which comprises:

-   -   (i) incubating a potential Class IIa HDAC inhibitor with a         protein which expresses the catalytic domain of Class IIa HDAC,         in a suitable assay buffer to maintain a pH of 6.0 to 8.5;     -   (ii) adding the substrate trifluoroacetylated lysine         (Boc-L-Lys(ε-trifluoroacetyl)-MCA) to produce a concentration of         1 μM and 100 μM in the sample; BIOMOL Fluor De Lys, Trypsin or         Lys-C; and a developer and incubating; and     -   (iii) stopping the incubation and determining the effect the         putative Class IIa HDAC inhibitor has on enzyme activity by         comparison with a standard.

EXAMPLE 1 The Deacetylating and Enzymatic Activity of HDAC4

The deacetylating activity of HDAC4, a class IIa histone deacetylase, was analysed by incubating different concentrations of C-terminally flagged HDAC4 (HDAC4-FLAG; 3-24 nM) with a histone substrate (histone H4-derived octamer peptide substrate containing a single acetylated lysine). FIG. 1 shows the activity at different concentrations as a percentage conversion of the acetylated substrate. The higher the concentration of the HDAC4-FLAG, the higher the deacetylating activity. The enzymatic activity of HDAC4-FLAG was measured in a conventional assay kit (Biomol) and with histone substrates by measuring its inhibition by Apicidin, MS27-275 and by LAQ824 (FIG. 2).

A cross-linking assay to analyze the binding of HDAC4-FLAG to the inhibitors was performed. The only compound able to significantly and specifically bind HDAC4 was LAQ824. Apicidin and MS25-275 were unable to bind the deacetylase, whilst showing good inhibition constants (in particular Apicidin with a low nanomolar IC₅₀) (FIG. 2). A possible explanation of this result could be that HDAC4-FLAG does not significantly contribute to the observed enzymatic activity, which rather reflects the involvement of one or more active endogenous co-purified HDACs. Infact, histone deacetylases expressed in mammalian cells interact with several endogenous HDACs.

To further analyze this point, the catalytic domain of HDAC4 (6xHis tagged) was expressed in E. coli. The HDAC4 C-terminal region from T-653 to L-1084, containing therefore the entire catalytic domain (HDAC4-CD), was produced, purified to homogeneity and the enzymatic activity analyzed with the histone-based (FIG. 3A) and the commercial Biomol HDAC assay (FIG. 3B; RFU, Fluorescence Units).

The conclusions which can be drawn from these experiments are that:

-   (a) HDAC4-CD enzymatic activity was about 100-fold lower (micromolar     amounts of enzyme) than that measured with flagged-HDAC4 in     transfected mammalian cells, thus suggesting that the pure enzyme is     a very weak deacetylase in the absence of interacting endogenous     enzymes; and -   (b) HDAC4-CD was resistant to the inhibitory effect of Apicidin, in     contrast with the sensitivity shown by flagged-HDAC4-associated     activity and in agreement with its incapability of binding this     compound

EXAMPLE 2 The Enzymatic Activity of HDACs 1, 3 and 8

A panel of HDAC inhibitors on class I HDACs 1, 3 and 8 was tested in an analogous manner to the HDAC4 (see Vannini et al., (2004) Proc. Natl. Acad. Sci. U.S.A., 101:15064-15069). The results showed that HDAC8 has a distinct inhibition pattern, which differs from that of HDAC1 and HDAC3. Whereas HDACs 1 and 3 were inhibited by all of the inhibitors, HDAC8 was inhibited not appreciably by Apicidin or MS27-275 (see FIG. 4). Thus, HDAC8 shows a more restricted inhibition pattern than other class I enzymes.

EXAMPLE 3 Preparation of tert-Butyl [(1S)-5-(trifluoroacetylamino)-1-[[(4-methyl-2-oxo-2H-1-benzopyran-6-yl) amino]carbonyl]pentyl]-carbamic Acid Ester (Trifluoroacetylated Lysine (Boc-L-Lys (ε-trifluoroacetyl)-MCA))

To a solution of Boc-Lys-AMC (1 equivalent, BACHEM: I-1880) in DCM and Et₃N (2 equivalents) at RT was added dropwise trifluoroacetic anhydride (1.2 equivalents). The resulting mixture was stirred at RT for 2 hours and was then diluted with DCM, washed with saturated aqueous NaHCO₃ solution and brine. The solution was dried (Na₂SO₄) and concentrated under reduced pressure whilst dry loading onto silica. The mixture was columned on silica eluting with 70-80% EtOAc/petroleum ether to yield the desired amide. ¹H NMR (300 MHz, d6-DMSO) δ: 10.40 (1H, br. S), 9.39 (1H, Br. S), 7.76 (1H, d, J=1.6 Hz), 7.72 (1H, d, J=8.4 Hz), 7.48 (1H, d, J=8.4 Hz), 7.10 (1H, d, J=7.6 Hz), 6.62 (1H, s), 4.10-4.00 (1H, m), 3.21-3.10 (2H, m), 2.38 (3H, s), 1.70-1.55 (2H, m), 1.54-1.43 (2H, m), 1.40-1.25 (2H, m), 1.37 (9H, s). MS (ES) C₂₃H₂₈F₃N₃O₆ requires: 499, found: 500 (M+H)⁺.

EXAMPLE 4 Comparing Activities on Boc-L-Lys(ε-trifluoroacetyl)-MCA with Activities on the Biomol Standard Substrate

The activities measured on Boc-L-Lys(ε-trifluoroacetyl)-MCA were compared with activities on the Biomol standard substrate (not shown). It was found that HDAC4-CD and HDAC8 work better on the Lys-Trifluoro substrate, HDAC3 works with the same efficiency and HDAC1 and HDAC6 did not work on this substrate (see FIG. 5).

An enzyme titration was performed on the HDAC4-CD enzyme, with a concentration of 0.5 nM being sufficient for the assay on the new substrate (data not shown) and which was about a thousand fold less than the amount needed for the commercial Biomol assay. The activity of HDAC4-CD (0.5 nM) on Lys-Trifluoro substrate is inhibited by LAQ824 and resistant to Apicidin, as shown in FIG. 6.

The results obtained on the Biomol substrate (HDAC4-CD=500 nM) are also shown in FIG. 6.

The experiments supra described with the Lys-Trifluoro substrate were done with the HDAC4catalytic domain (HDAC4-CD). To test the behavior of the complete protein, the full length HDAC4protein (HDAC4-FLAG) was also analysed. Other class IIa HDACs, namely HDAC 5 and 7, were also tested, together with HDAC1, HDAC3 and HDAC8 as controls. After transfection in mammalian cells, the proteins were purified and analyzed. In these conditions associated HDACs did not significantly contribute to the activity, since very low (nanomolar) amounts of enzyme were used in the assay. Results are shown in FIG. 7 and FIG. 8.

EXAMPLE 5 HDAC4 Assay

The following assay was used to screen for potential HDAC4 inhibitors and measure their activity.

HDAC 4 Expression and Affinity Purification

The His-tagged HDAC 4, wild-type catalytic domain, was expressed in E. coli strain BL21 Star™ (DE3). The cells were grown at 37° C. in minimum medium supplemented with 1 g/l (¹⁵NH₄)₂SO₄ and 5 g/l glucose, and 100 μM of ZnCl₂ to an optical density of 0.8 at 600 nm and induced with IPTG for 16 hr at 23° C. At 23° C. more than 80% of the protein was soluble.

Bacterial pellets were resuspended in 25 mM Hepes pH 7.5, 200 mM KCl, 0.5% NP-40, 20% glycerol 1 mM DTT and supplemented with Complete EDTA-free protease inhibitor. Subsequently bacterial pellets were lysed by microfluidizer, and centrifuged at 15000 rpm for 30 min.

The soluble fraction was diluted 1:1 with 25mM Hepes pH 7.5, 200 mM KCl, 1 mM DTT and was loaded directly on His Trap HP 5 ml (Amersham Biosciences). The protein was eluted at 200 mM Imidazole. The fractions with HDAC 4 were diluted 1:3 with 25 mM Hepes pH 7.5, 5% glycerol, 0.1% of NP-40, 1 mM DTT. Then the solution was loaded on a Resource Q equilibrated with 25 mM Hepes pH 7.5, 10% glycerol, 50 mM KCl, 0.1% of NP-40, 1 mM DTT. HDAC 4 was eluted with a salt gradient (0-250) mM of KCl. The product was fractionated by preparative SEC (G-75, Superdex 75 26/60 Amersham Biosciences) (25 mM Hepes pH 7.5, 150 mM KCl, 0.1% of β-octyl glucopiranoside, 1 mM DTT) to give the final product. Analytical SEC indicated that this product was monomeric. The protein was concentrated at ≈100 μM.

HDAC 4 Assay Working Reagents

-   TSA Stock: TSA is provided as a 10 mM solution in 100% DMSO. -   Assay buffer: 25 mM Tris/HCl pH8, 137 mM NaCl, 2.7 mM KCl, 1 mM     MgCl₂, 0.1 mg/ml BSA -   Diluted substrate solution: tert-butyl     {(1S)-1-{[(4-methyl-2-oxo-2H-chromen-7-yl)amino]carbonyl}-5-[(trifluoroacetyl)     amino]pentyl} carbamate (trifluoroacetylated lysine     (Boc-L-Lys(ε-trifluoroacetyl)-MCA)) is diluted to 200 μM with Tris 1     mM pH 7.4 prior to each use. The final concentration in the assay is     20 μM. -   Diluted developer solution: The commercial 20X developer concentrate     (KI-105, BioMol Research Laboratories) is diluted 1:167 into Tris 1     mM pH7.4. 2 μM [final] TSA to this solution increases its ability to     stop the reaction. -   Enzyme working solution: Enzyme is diluted in 1.25× assay buffer     prior to each use from a fresh aliquot of enzyme. The final     concentration in the assay is 0.2 nM.

Experimental Design:

The reaction is performed in 96-well microplate in a final volume of 50 μl/well. Add 5 μl of DMSO/compound solution, add 40 μl of HDAC 4 enzyme in assay buffer and incubate 10′ at RT. Start the reaction by adding 5 μl of the 200 μM substrate solution and incubate 1 hr at 37° C. Stop the reaction by adding 50 μl of developer/4 μM TSA solution and incubate 30 min at RT. Measure the fluorescence at ex.360 nM and em.460 nM. 

1. (canceled)
 2. (canceled)
 3. An assay for determining the concentration of a Class IIa HDAC in a sample which comprises: (i) incubating said sample suspected of containing said Class IIa HDAC with trifluoroacetylated lysine (Boc-L-Lys(ε-trifluoroacetyl)-MCA); (ii) adding a suitable enzyme capable of cleaving trifluoroacetylated lysine (Boc-L-Lys(ε-trifluoroacetyl)-MCA) developer and (iii) measuring the concentration of said enzyme as a determinant of the concentration of said Class IIa HDAC in said sample.
 4. The assay for detecting Class IIa HDAC in a test sample, comprising the steps of: (i) incubating said test sample potentially containing Class IIa HDAC with suitable amounts of trifluoroacetylated lysine (Boc-L-Lys (ε-trifluoroacetyl)-MCA); and (ii) detecting the presence of MCA by its fluorescence.
 5. The assay for identifying a potential Class IIa HDAC inhibitor, which comprises: (i) incubating a potential Class IIa HDAC inhibitor with nucelci acid molecule under conditions favoring expression a protein comprising a catalytic domain of Class IIa HDAC; (ii) adding suitable amounts of trifluoroacetylated lysine (Boc-L-Lys(ε-trifluoroacetyl)-MCA) and an enzyme under conditions favoring cleavage of said trifluoroacetylated lysine (Boc-L-Lys(ε-trifluoroacetyl)-MCA); and (iii) determining concentration of a cleavage product as a determinant of an HDAC inhibitor relative to a control sample that does not contain said inhibitor.
 6. The assay according to claim 5 wherein said enzyme is selected from the group consisting of BIOMOL Fluor De Lys, Trypsin and Lys-C.
 7. The assay according to claim 5 wherein said Class IIa HDAC enzyme is present at a concentration of 0.05-50 nM.
 8. The assay according to claim 7 wherein the assay is performed at a pH value between 6.0 and 8.5.
 9. The assay according to claim 8 further comprising monovalent and/or divalent cations.
 10. The assay according to claim 9 further comprising bovine serum albumin.
 11. The assay according to claim 10 wherein the substrate trifluoroacetylated lysine (Boc-L-Lys(ε-trifluoroacetyl)-MCA) is added at a concentration range between 1 μM and 100 μM.
 12. The assay according to claim 11 wherein the protein which expresses the catalytic domain of Class IIa HDAC is E coli.
 13. The assay according to claim 12 wherein the Class IIa HDACs is HDAC4. 